Hybrid compositions for intracellular targeting

ABSTRACT

Hybrid compounds comprising a first domain and a second domain are provided. The first domain and the second domain are preferably covalently linked, and the first domain comprises a domain which is capable of specific binding to Gb 3 ; and the second domain comprising a moiety selected from the group consisting of drug moiety, a nucleic acid, a probe, a polypeptide, and a hook, with the proviso that the second domain is not a verotoxin or a fragment thereof. Mehtods of preapring and using the hybrid compounds are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. 119(e) toco-pending U.S. provisional application Serial No. 60/031,668, filedNov. 22, 1996; No. 60/061,050, filed Oct. 3, 1997; and No. 60/061,044,filed Oct. 4, 1997; the contents of all of which are hereby incorporatedby reference.

BACKGROUND OF THE INVENTION

[0002] Recent advances in the understanding of the molecular bases ofdisease states and conditions have permitted the rationally-baseddevelopment, at least in principle, of therapies which are specificallydesigned to target a particular molecular entity or entities.Unfortunately, a practical difficulty often arises in attempting totreat diseases with rationally-designed drugs, viz., while the drug maywork as expected in vitro, in order to have the desired therapeuticeffect the drug must be able to reach the site of action in vivo withoutbeing metabolically inactivated or degraded. In the case of drugs whichmust reach an intracellular site to be effective, providing the drug ina form capable of reaching the desired site can be difficult. Althoughmany proposals have been made to deal with this problem, there are fewapproaches which are broadly applicable to a wide variety of drugs anddisease states. One approach has been to administer the drug in a form,such as a liposome preparation, which allows the drug to cross the cellmembrane. However, non-targeted liposomes may deliver the drug tonon-target cells or organs, and the use of specifically-targetedliposomes can be expensive or inconvenient.

[0003] The endocytotic pathway of many protein toxins comprisingseparate A (enzymatic) and B (receptor binding) subunits, involves cellbinding, internalization, translocation from an intracellularcompartment to the cytosol, and enzymatic inactivation of theirintracellular targets (1, 2). After translocation to the cytoplasm, theA subunits of ricin, abrin, modeccin, and verotoxins catalyticallyinactivate the 28 S RNA of 60 S ribosomal subunits, leading to aninhibition of cellular protein synthesis (3, 4). In addition, both theholotoxin and the B subunit are capable of inducing programmed celldeath (apoptosis) (5-7).

[0004] The E coli derived family of verotoxins (or Shiga-like toxins)comprise VT1, VT2 and VT2c, which are involved in the etiology ofmicrovascular disease in man (8), primarily in the very young andelderly (9), and VT2e which causes edema disease in pigs (10). Theglycolipid globotriaosylceramide (gala1-4galb1-4glc cer.-Gb₃) at theplasma membrane is the specific receptor for all verotoxins and mediatesthe internalization of verotoxin (VT1) into susceptible cells by cappingand receptor-mediated endocytosis (RME) (11). Verotoxin is the onlyglycolipid binding ligand that is internalized into eukaryotic cells bymeans of RME (12-14). In addition to receptor concentration, bothheterogeneous fatty acid composition of Gb₃ (15, 16) and phospholipidchain length within the phospholipid bilayer (17) play important rolesin binding and internalization of VT. Molecular modeling studies of theGb₃ binding site in the B subunit (18) show that different conformers ofmembrane Gb₃ may bind in different sites. Such conformers may be relatedto the Gb₃ fatty acid content and membrane phospholipid microenvironment(18-20).

[0005] The requirement for retrograde transport for intoxication ofcells by verotoxin was first demonstrated by Sandvig (21). A431 cellsare resistant to VT. These cells expressed Gb₃ but the toxinreceptor-complex was internalized to endosomes and lysosomes. However,following growth in the presence of butyric acid, an inducer of celldifferentiation, A431 cells became VT-sensitive, coincident with thedetection of internalized toxin in Golgi cisternae, ER and even in thenuclear envelope (21). Similar targeting of both the holotoxin and Bsubunit to the nuclear envelope in highly toxin sensitive B lymphomashas been found (11).

[0006] In studying the sensitivity of human astrocytoma cell lines toverotoxin, significant differences which do not correlate with the levelof receptor expression (6). Similarly, multiple drug resistant (MDR)variants of ovarian tumor cells lines were hypersensitive to VT ascompared to the parental cell line, without major increase in receptorexpression (22). Based on these discrepancies, Gb₃-dependentintracellular traffic plays a major role in determining cell sensitivityto VT.

SUMMARY OF THE INVENTION

[0007] The invention relates to hybrid compounds, and methods ofpreparing and using the same.

[0008] In one aspect, the invention provides a hybrid moleculecomprising a first domain and a second domain covalently linked, wherein(a) said first domain comprises a domain which is capable of specificbinding to Gb₃; (b) said second domair comprising a moiety selected fromthe group consisting of drug moiety, a nucleic acid, a probe, apolypeptide, and a hook, with the proviso that the second domain is nota verotoxin or a fragment thereof. In preferred embodiments, the firstdomain is a verotoxin or a verotoxin subunit; the first domain is VT-B;the second domain is a polypeptide; the polypeptide is a DNA bindingelement; the second domain is a nucleic acid; the nucleic acid is anantisense nucleic acid. In another aspect, the invention provides apharmaceutical composition comprising a hybrid molecule of the inventionand a pharmaceutically acceptable carrier.

[0009] In another aspect, the invention provides a method for modulatinga cell-associated activity comprising contacting a cell with the hybridmolecule of the invention such that a cell associated activity isaltered relative to the cell-associated activity of the cell in theabsence of the hybrid molecule.

[0010] In another aspect, the invention relates to a method fordirecting the delivery of the hybrid compound of the invention to aparticular intracellular location in a cell, the method comprisingcontacting the cell with the hybrid compound, optionally in the presenceof a compound which alters fatty acid composition of Gb₃, such that thehybrid compound is delivered to a particular intracellular location inthe cell.

[0011] In another aspect, the invention provides a se of a hybridcompound of the invention for the manufacture of a medicament fortreatment, prophylaxis, or diagnosis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 VT1 cytotoxicity in different human astrocytoma cell linesVT1 cytotoxicity for six astrocytoma cell lines was determined asdescribed in the Methods. SF-539 is the most, and XF-498 is the leastsensitive of the cells to VT1 cytotoxicity. Each value represents themean±S.D. of triplicates. This experiment was repeated three times withsimilar results.

[0013]FIG. 2 Astrocytoma cell surface 125 I VT1 binding SF 529 (▪), XF498 (◯) and butyrate treated XF 498 (Δ) cells were treated with serialdilutions of ¹²⁵I-VT1 in PBS at 4° C., incubated for 1 h and binding wasdetermined as described in the Methods. All three cell lines demonstratesimilar level of VT1 binding. Each value represents the mean±S.D. oftriplicate determinations.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The invention relates to hybrid compounds, nucleic acidsencodingg the hybrid molecules, to methods of preparing the hybridcompounds and nucleic acids which encode them, and to methods oftreating subjects with the hybrid compositions.

[0015] In one aspect, the invention provides a hybrid compounds. Thehybrid compound includes a first domain and a second domain; the firstand second domains are, preferably, covalently linked. The first domainis a binding domain capable of specific binding to globotriaosylceramide(Gb₃) and being internalized into a cell which expresses Gb₃ on the cellsurface. The second domain is a functional domain which includes amolecular moiety which is to be delivered into the cell, e.g., to thecell nucleus. The second domain is preferably not a verotoxin, averotoxin subunit, or a fragment thereof. The second domain can be, forexample, a drug moiety (e.g., a drug molecule bound to the firstdomain), a nucleic acid (e.g., a gene which encodes an exogenousprotein, or a nucleic acid which regulates gene expression in a cell,such as antisense nucleic acid, repressors, or trans activators), aprobe (such as a fluorescent probe), a protein, and the like. The seconddomain can also be a domain which functions as a handle or hook forcomplexation or binding to another moiety or moieties. For example, thesecond domain can be a member of a specific binding pair (such asbiotin/streptavidin, hormone/receptor, binding protein/ligand, and thelike), which can be complexed with or bound to the other member of thespecific binding pair, which can, in turn, be bound to a moiety which isdesired to be delivered into the cell.

[0016] A hybrid molecule of the invention can be a fusion protein of thefirst domain (e.g., verotoxin B subunit (VT-B)) with a proteinaceoussecond domain (such as a DNA binding protein. The fusion protein can, incertain embodiments, be bound to or complexed with, a moiety to bedelivered into a cell. For example, in the example of a VT-B/DNA bindingprotein fusion (see, e.g., Example 4, infra), the fusion protein canmade, optionally purified, and then be bound to a nucleic acid to form acomplex of the hybrid compound with the nucleic acid. Upon presentationof the fusion protein/nucleic acid complex to a cell, and endocytosis ofthe fusion protein, with the associated nucleic acid, into the cell, thenucleic acid is delivered into the cell, for example to the nucleus,e.g., to promote transfection of the cell with the nucleic acid.

[0017] A hybrid molecule of the invention can also include a non-fusioncovalent adduct of the first domain and the second domain. For examples,methods for covalent modification of proteins are well known, and kitsfor accomplishing the covalent conjugation of proteins to other proteinand non-protein moieties are commercially available. For example, apolypeptide or protein such as VT-B can be linked through the use ofheterobifunctional linking agents to moieties such as other proteins,nucleic acids, drugs, probes or labels, and the like. As an example, afirst domain, such as VT-B can be covalently linked to a polypeptidesuch as polylysine (see, e.g., Example 5, infra). Polylysine can complexwith nucleic acids such as DNA for delivery of the nucleic acid to acell (see, e.g., U.S. Pat. Nos. 5,635,383 and 5,166,320 to Wu); thus, aVT-B/polylysine conjugate can be used to bind to a nucleic acid andtransport the nucleic acid into a cell, e.g., for applications such asgene therapy, anti-sense therapy, and the like.

[0018] In another embodiment, the invention relates to use of a hybridcompound of the invention for in therapy, prophylaxis, or diagnosis,e.g., use of a hybrid compound of the invention for the preparation of amedicament for treatment, prophylaxis, or diagnosis. The hybridcompounds of the invention find use in methods treatments such astreatment of cancer, e.g., by methods such as gene therapy (i.e., byintroduction of a gene encoding an exogenous protein into a cell, orintroduction of a suppressor or trans activator sequence into a cell),antisense nucleic acid therapy (i.e., by introduction of antisensesequences, e.g., antisense to an oncogene of a cancerous cell);targeting of a chemotherapeutic drug to a cell, and the like.

[0019] As described in more detail hereinbelow, the invention furtherprovides methods for altering the intracellular target of a hybridcompound of the invention. Thus, for example, treatment of a cell withsodium butyrate can alter the intracellular destination of VT-B, andtherefore a hybrid compound of the invention. As described infra, incertain cells, VT-B is normally transported to components of the Golgiapparatus in the absence of sodium butyrate; however, in the presence ofbutyrate, VT-B is transported to elements of the endoplasmic reticulumand/or the nuclear envelope. As is also described in more detailhereinbelow, this effect is believed to be due, at least in part, toalterations in the fatty acid composition of the cell surface glycolipidGb₃. The invention contemplates the selective transport of a hybridcompound of the invention to a selected location in the cell, e.g., thenuclear membrane or nucleus. This feature of the invention is especiallyuseful for gene therapy applications (or antisense treatments) in whichthe nuclear genome, rather than the DNA in the cytoplasm or cytoplasmicorganelles, is to be targeted. Accordingly, in certain embodiments, theinvention contemplates treatment of a cell with a compound (e.g.,butyrate), or under conditions, capable of effecting a change in thefatty acid composition of Gb3, and thereby promoting selective transportof a hybrid compound of the invention to a pre-selected intracellularlocation (e.g., the nucleus).

[0020] It will be appreciated by the skilled artisan that use of thehybrid compounds of the invention provides benefits such as specificityof cell targeting, the ability to deliver almost any therapeutic moietyto a cell, the ability to specifically target subcellular structures,and the like. For example, it is well known that certain cell types,including certain cancer cells, express large amounts of Gb₃ on the cellsurface, while other cell types express little Gb₃ at the cell surface.The hybrid compounds of the invention will be readily endocytosed by theformer cell types, but will be less readily transported into the lattercell type. Thus, a cell type which express large amounts of Gb₃ on thecell surface can be selectively targeted using the hybrid compounds ofthe invention. Importantly, certain cancer cells show greater Gb₃ levelsthan normal cells; for example, primary ovarian tumor cells have beenfound to show greater levels of Gb₃ than normal ovarian tissue cells.Thus, the hybrid compounds of the invention can be used to deliver adrug, a gene, or another therapeutic agent specifically to cancerouscell types while sparing normal tissues.

[0021] The First Domain

[0022] The first domain of the hybrid compounds of the inventioncomprises a domain which is capable of specific binding toglobotriaosylceramide (Gb₃), and is capable of being internalized into acell which expresses Gb₃ on the cell surface; such a domain will forconvenience sometimes be referred to herein as a “VT binding domain”although, as described herein, first domains suitable for use in theinvention are not limited to verotoxins or fragments thereof. Domainssuitable for use as a first domain of a hybrid compound of the inventioninclude native verotoxins (VTs), subunits of verotoxins (e.g., VT-Bsubunit) which bind to Gb₃, and polypeptides comprising amino acidsequences homologous to and/or derived from the amino acid sequence of anative VT binding domain, which can include more, fewer (e.g., adeletion or truncation), or an equal number (e.g., point mutations) ofamino acids than a full length VT binding domain protein, whileretaining substantial specific binding affinity for Gb₃ (or Burkitt'slymphoma associated antigen (BLA) (Nudelman, et al. Science 220: 509(1983), also known as the B-cell differentiation antigen CD77). Thus, a“VT binding domain”, as used herein, refers to the Gb₃ receptor bindingsubunit of verotoxins or homologous domains which have Gb₃ bindingactivity. It will be appreciated that certain proteins or polypeptidesare known which have substantial homology to verotoxin binding domains(e.g., CD19, a 95 kDa immunoglobulin superfamily integral membraneglycoprotein present on the cell surface of human B lymphocytes from theearly stage of B-cell development to the terminal differentiation ofB-cells to plasma cells (Nadler, et al. J. Immunol. 131: 244-250 (1983);Lingwood, C. A. (1996) Trends in Microbiol. 4(4):147-153; Maloney, M. D.and Lingwood, C. A. (1994) J. Exp. Med. 180:191-201; Nyholm, P. G.,Magnusson, G. and Lingwood, C. (1996) Chem. Biol. 3:263-275) and canbind to Gb₃ or Gb₃-like cell surface moieties; use of such homologousproteins or polypeptides is contemplated in the hybrid compounds of theinvention. In one embodiment, the first domain of a hybrid compound ofthe invention is at least about 30%, 40%, more preferably at least about50%, 60%, even more preferably at least about 70%, 80%, yet even morepreferably at least about 90%, and most preferably at least about 95%(or more) homologous to a Gb₃ binding domain of a native verotoxin (orverotoxin subunit). Typically, biologically active portions comprise adomain or motif with at least one activity of a VT binding domain. Abiologically active portion of a VT binding domain protein can be apolypeptide which is, for example, 10, 25, 50, 100 or more amino acidsin length.

[0023] In one embodiment, a biologically active portion of a VT bindingdomain protein comprises at least one Gb₃ binding domain. In otherembodiments, a biologically active portion of a VT binding domainprotein comprises two, three or four Gb₃ binding domains.

[0024] Moreover, other biologically active portions, in which otherregions of the protein are deleted, can be prepared by recombinanttechniques and evaluated for one or more of the functional activities ofa native VT binding domain protein. It will be appreciated by theskilled artisan that VT holotoxin has significant toxicity to certaincell types. The toxicity of VT holotoxin is largely (although notentirely) due to the A subunit of VT; isolated VT-B has much lowertoxicity to most cell types than does the holotoxin. Accordingly, inpreferred embodiments, a hybrid compound of the invention does notcomprise VT subunit, or portion thereof, which confers cell toxicity onVT holotoxin; e.g., in preferred embodiments, a hybrid compound of theinvention does not include VT A subunit, or any substantially cytotoxicportion thereof. Thus, in a preferred embodiment, the first domain of ahybrid compound of the invention consists essentially of VT-B, or ahomolog thereof, or a fragment thereof, which is substantially free ofVT A subunit or portions of VT A subunit. Of course, if it desired touse a hybrid compound of the invention to kill a cell (such as a cancercell or a virally-infected cell), a toxic moiety can be employed as thesecond domain, e.g., all or part of a toxic protein such as ricin,diphtheria toxin, tetanus toxin, and the like.

[0025] To determine the percent homology of two amino acid sequences orof two nucleic acids, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoor nucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are homologous at that position(i.e., as used herein amino acid or nucleic acid “homology” isequivalent to amino acid or nucleic acid “identity”). The percenthomology between the two sequences is a function of the number ofidentical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100).

[0026] The Second Domain

[0027] The second domain of a hybrid compound of the invention can bealmost any moiety which is desired to be transported into a cell (eitherin vitro or in vivo) and which is capable of being bound (e.g., througha covalent bond) to the first domain of the compound of the invention.The second domain also can be a handle or hook for binding or complexingto a third component (e.g., a nucleic acid, and the like).

[0028] Examples of second domains contemplated for use in the hybridcompounds of the invention include proteins, polypeptides, and fragmentsthereof (including peptidomimetics, amino acid analogs, and the like).For example, a second domain can comprise a protein (e.g.,erythropoeitin, human growth hormone, insulin, somatostatin, EGF, andInterleukins I, II,III, IV and VI, and the like. It will be appreciatedthat the use of a second domain which itself has a particularspecificaty for a cell surface receptor can provide additionalspecificity to the hybrid compound of the invention when administered toa subject, e.g., a human or animal. For example, a hybrid compound whichinclude VT-B as a first domain and substance P as a second domain maypreferentially target cells which express both Gb₃ and substance Preceptors.

[0029] Additional examples of second domains include nucleic acids,e.g., DNA, RNA, DNA/RNA chimeric nucleic acids, and the like, as well asanalogs of nucleic acids such as phosphorothioate nucleic acids (see,e.g., Cornish et al., Pharmacol. Com. 3: 239-247, 1993; Crooke, Ann.Rev. Pharm. Toxicol. 32: 329-376, 1992; Iversen, Anti-Cancer Drug Design6: 531-538, 1991). A nucleic acid can, e.g., encode a protein orfragment thereof, can be a regulatory sequence such as a repressor orpromoter sequence, or can be a complement to a nucleotide sequencepresent in a cell, e.g., for use in antisense therapy.

[0030] Still further examples of second domains useful in the hybridcompounds of the invention include hormones (e.g., steroids) or otherbiologically active moieties (e.g., retinoids).

[0031] Yet further examples of second domains include probes, e.g.,probes for examining cell structure (e.g., for use in vitro, includingradioisotope labels, fluorescent labels, heavy atom labels such as goldparticles, and the like), labels for in vivo studies (e.g., labelsdetectable by X-ray, magnetic resonance imaging, and the like). Thus,the invention provides hybrid compounds which find use as diagnosticagents, e.g., for use in cell culture studies or when formulated in apharmaceutically acceptable vehicle for administration to a subject.

[0032] Still other examples of second domains include a handle or hookfor binding or complexing to a third component. For example, a secondmoiety can be a member of a specific binding pair (e.g.,receptor/ligand, hormone/receptor, nucleic acid/complement,enzyme/ligand, and the like). A hybrid compound which includes a hook asa second domain can then be used to transport a complementary moleculeinto a cell by endocytosis, e.g., a hybrid compound which includes astreptavidin sequence (or portion thereof) can be used to bind tobiotin, which in turn can be bound or coupled to a moiety for deliveryinto a cell (e.g., a biotinylated nucleic acid, or the like). A secondmoiety can also be, e.g., a moiety which binds non-specifically to athird component which is to be delivered to a cell. For example, aVT-B/polycation (e.g., polylysine) hybrid compound (see, e.g., Example5, infra) can be used to deliver a negatively-charged compound (e.g., anucleic acid such as DNA) to a cell for endocytosis.

[0033] Hybrid Compounds

[0034] The invention thus contemplates a wide variety of hybridcompounds, which have a great number of uses.

[0035] In certain embodiments, the hybrid compounds of the inventioninclude a first domain and a second domain (e.g., as described above),covalently linked. The covalent linkage can be provided in many ways,which will be routine to the ordinarily skilled artisan. For example,the second domain can be covalently bound to the first domain throughchemistry known for the covalent modification of proteins, e.g., the useof heterobifunctional linkers, in which one functional group can reactwith a functional group of a protein (e.g., the first domain) (e.g., aside chain thiol, amine, or carboxylate of the first domain) and asecond functional group which can react with a moiety of the seconddomain (e.g., a side chain group where the second domain is apolypeptide, a hydroxyl group of a nucleic acid, a drug, or a hormone,and the like). A wide variety of bifunctional or polyfunctionalcross-linking reagents, both homo- and heterofunctional, are known inthe art and are commercially available (e.g., Pierce Chemical Co.,Rockford, Ill.). accordingly, one of ordinary skill in the art will beable to prepare a wide variety of hybrid compounds of the inventionusing no more than routine experimentation.

[0036] The invention also provides fusion proteins, e.g., VT bindingdomain chimeric or fusion proteins. The term “fusion protein” isintended to describe at least two polypeptides, typically from differentsources, which are operatively linked. With regard to the polypeptides,the term “operatively linked” is intended to mean that the twopolypeptides are connected in manner such that each polypeptide canserve its intended function. Typically (although not exclusively), thetwo polypeptides are covalently attached through peptide bonds. Thefusion protein is preferably produced by standard recombinant DNAtechniques. For example, a DNA molecule encoding the first polypeptideis ligated to another DNA molecule encoding the second polypeptide, andthe resultant hybrid DNA molecule is expressed in a host cell to producethe fusion protein. The DNA molecules are ligated to each other in a 5′to 3′ orientation such that, after ligation, the translational frame ofthe encoded polypeptides is not altered (i.e., the DNA molecules areligated to each other in-frame).

[0037] Thus, a VT binding domain “chimeric protein” or “fusion protein”comprises a first domain, e.g., a VT binding domain polypeptideoperatively linked to a second domain, e.g., a second polypeptide, whichpreferably is a non-VT binding domain polypeptide, e.g., a polypeptidewhich is not substantially homologous to a VT binding domain protein,e.g., a protein which is different from the VT binding domain proteinand which is derived from the same or a different organism. The seconddomain polypeptide can be fused to the N-terminus or C-terminus of thefirst domain polypeptide. In a preferred embodiment, the second domaincomprises at least about 5 amino acid residues, more preferably about10, 20, 30, 40, 50, 100 or 200 amino acid residues.

[0038] For example, in one embodiment a VT binding domain fusion proteincomprises a Gb₃ binding domain operably linked to the extracellulardomain of a second protein. Such fusion proteins can be further utilizedin delivering molecules to the intracellular region of a cell, e.g.,delivery of pharmaceutical compositions.

[0039] In yet another embodiment, the fusion protein is a GST-VT bindingdomain fusion protein in which the VT binding domain sequences are fusedto the C-terminus of the GST sequences. Such fusion proteins canfacilitate the purification of recombinant VT binding domain.

[0040] Preferably, a VT binding domain chimeric or fusion protein of theinvention is produced by standard recombinant DNA techniques. Forexample, DNA fragments coding for the different polypeptide sequencesare ligated together in-frame in accordance with conventionaltechniques, for example by employing blunt-ended or stagger-endedtermini for ligation, restriction enzyme digestion to provide forappropriate termini, filling-in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining, andenzymatic ligation. In another embodiment, the fusion gene can besynthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhargs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Current Protocols in Molecular Biology, eds. Ausubel et al.John Wiley & Sons: 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A VT binding domain-encoding nucleic acid can be clonedinto such an expression vector such that the fusion moiety is linkedin-frame to the VT binding domain protein.

[0041] It will be appreciated from the foregoing that the invention alsoprovides nucleic acids (e.g., DNA) which encode the fusion proteins ofthe invention. Nucleic acids which encode the fusion proteins of theinvention are nucleic acids of the invention.

[0042] In one embodiment, variants of the VT binding domain portion ofthe VT binding domain fusion protein which function as either VT bindingdomain agonists (mimetics) or as VT binding domain antagonists can beidentified by screening combinatorial libraries of mutants, e.g.,truncation mutants, of the VT binding domain protein for VT bindingdomain protein agonist or antagonist activity. In one embodiment, avariegated library of VT binding domain variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of VT binding domainvariants can be produced by, for example, enzymatically ligating amixture of synthetic oligonucleotides into gene sequences such that adegenerate set of potential VT binding domain sequences is expressibleas individual polypeptides, or alternatively, as a set of larger fusionproteins (e.g., for phage display) containing the set of VT bindingdomain sequences therein. There are a variety of methods which can beused to produce libraries of potential VT binding domain variants from adegenerate oligonucleotide sequence. Chemical synthesis of a degenerategene sequence can be performed in an automatic DNA synthesizer, and thesynthetic gene then ligated into an appropriate expression vector. Useof a degenerate set of genes allows for the provision, in one mixture,of all of the sequences encoding the desired set of potential VT bindingdomain sequences. Methods for synthesizing degenerate oligonucleotidesare known in the art (see, e.g., Narang, S. A. (1983) Tetrahedron 39:3;Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984)Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.

[0043] In addition, libraries of fragments of the VT binding domainprotein coding sequence can be used to generate a variegated populationof VT binding domain fragments for screening and subsequent selection ofvariants of a VT binding domain protein. In one embodiment, a library ofcoding sequence fragments can be generated by treating a double strandedPCR fragment of a VT binding domain coding sequence with a nucleaseunder conditions wherein nicking occurs only about once per molecule,denaturing the double stranded DNA, renaturing the DNA to form doublestranded DNA which can include sense/antisense pairs from differentnicked products, removing single stranded portions from reformedduplexes by treatment with S1 nuclease, and ligating the resultingfragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal and internalfragments of various sizes of the VT binding domain protein.

[0044] Several techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having aselected property. Such techniques are adaptable for rapid screening ofthe gene libraries generated by the combinatorial mutagenesis of VTbinding domain proteins. The most widely used techniques, which areamenable to high through-put analysis, for screening large genelibraries typically include cloning the gene library into replicableexpression vectors, transforming appropriate cells with the resultinglibrary of vectors, and expressing the combinatorial genes underconditions in which detection of a desired activity facilitatesisolation of the vector encoding the gene whose product was detected.Recrusive ensemble mutagenesis (REM), a new technique which enhances thefrequency of functional mutants in the libraries, can be used incombination with the screening assays to identify VT binding domainvariants (Arkin and Yourvan (1992) PNAS 89:7811-7815; Delgrave et al.(1993) Protein Engineering 6(3):327-331).

[0045] An isolated VT binding domain protein, or a portion or fragmentthereof, can be used as an immunogen to generate antibodies that bind VTbinding domain using standard techniques for polyclonal and monoclonalantibody preparation. The full-length VT binding domain protein can beused or, alternatively, the invention provides antigenic peptidefragments of VT binding domain for use as immunogens. The antigenicpeptide of VT binding domain comprises at least 8 amino acid residues ofthe amino acid sequence of VT binding domain such that an antibodyraised against the peptide forms a specific immune complex with VTbinding domain. Preferably, the antigenic peptide comprises at least 10amino acid residues, more preferably at least 15 amino acid residues,even more preferably at least 20 amino acid residues, and mostpreferably at least 30 amino acid residues.

[0046] An antibody according to the invention can be used to deliver amoiety into a cell, e.g., a moiety can be bound to an antibody, and theantibody can be bound to a VT binding domain. The complex can then beinternalized into a cell as described herein, thereby delivering themoiety to the cell.

[0047] A VT binding domain immunogen typically is used to prepareantibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouseor other mammal) with the immunogen. An appropriate immunogenicpreparation can contain, for example, recombinantly expressed VT bindingdomain protein or a chemically synthesized VT binding domainpolypeptide. The preparation can further include an adjuvant, such asFreund's complete or incomplete adjuvant, or similar immunostimulatoryagent. Immunization of a suitable subject with an immunogenic VT bindingdomain preparation induces a polyclonal anti-VT binding domain antibodyresponse.

[0048] Accordingly, another aspect of the invention pertains to anti-VTbinding domain antibodies. The term “antibody” as used herein refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds (immunoreacts with) an antigen,such as VT binding domain. Examples of immunologically active portionsof immunoglobulin molecules include F(ab) and F(ab′)₂ fragments whichcan be generated by treating the antibody with an enzyme such as pepsin.The invention provides polyclonal and monoclonal antibodies that bind VTbinding domain. The term “monoclonal antibody” or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one species of an antigen binding sitecapable of immunoreacting with a particular epitope of VT bindingdomain. A monoclonal antibody composition thus typically displays asingle binding affinity for a particular VT binding domain protein withwhich it immunoreacts.

[0049] Polyclonal anti-VT binding domain antibodies can be prepared asdescribed above by immunizing a suitable subject with a VT bindingdomain immunogen. The anti-VT binding domain antibody titer in theimmunized subject can be monitored over time by standard techniques,such as with an enzyme linked immunosorbent assay (ELISA) usingimmobilized VT binding domain. If desired, the antibody moleculesdirected against VT binding domain can be isolated from the mammal(e.g., from the blood) and further purified by well known techniques,such as protein A chromatography to obtain the IgG fraction. At anappropriate time after immunization, e.g., when the anti-VT bindingdomain antibody titers are highest, antibody-producing cells can beobtained from the subject and used to prepare monoclonal antibodies bystandard techniques, such as the hybridoma technique originallydescribed by Kohler and Milstein (1975) Nature 256:495-497) (see also,Brown et al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol.Chem. 255:4980-83; Yeh et al. (1976) PNAS 76:2927-31; and Yeh et al.(1982) Int. J. Cancer 29:269-75), the more recent human B cell hybridomatechnique (Kozbor et al. (1983) Immunol Today 4:72), the EBV-hybridomatechnique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology forproducing monoclonal antibody hybridomas is well known (see generally R.H. Kenneth, in Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lemer(1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977)Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typicallya myeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with a VT binding domain immunogen as described above, and theculture supernatants of the resulting hybridoma cells are screened toidentify a hybridoma producing a monoclonal antibody that binds VTbinding domain.

[0050] Any of the many well known protocols used for fusing lymphocytesand immortalized cell lines can be applied for the purpose of generatingan anti-VT binding domain monoclonal antibody (see, e.g., G. Galfre etal. (1977) Nature 266:55052; Gefter et al. Somatic Cell Genet., citedsupra; Lemer, Yale J. Biol. Med., cited supra; Kenneth, MonoclonalAntibodies, cited supra). Moreover, the ordinarily skilled worker willappreciate that there are many variations of such methods which alsowould be useful. Typically, the immortal cell line (e.g., a myeloma cellline) is derived from the same mammalian species as the lymphocytes. Forexample, murine hybridomas can be made by fusing lymphocytes from amouse immunized with an immunogenic preparation of the present inventionwith an immortalized mouse cell line. Preferred immortal cell lines aremouse myeloma cell lines that are sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a numberof myeloma cell lines can be used as a fusion partner according tostandard techniques, e.g., the P3-NS1/1-Ag4-4, P3-x63-Ag8.653 orSp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC.Typically, HAT-sensitive mouse myeloma cells are fused to mousesplenocytes using polyethylene glycol (“PEG”). Hybridoma cells resultingfrom the fusion are then selected using HAT medium, which kills unfusedand unproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bind VTbinding domain, e.g., using a standard ELISA assay.

[0051] Alternative to preparing monoclonal antibody-secretinghybridomas, a monoclonal anti-VT binding domain antibody can beidentified and isolated by screening a recombinant combinatorialimmunoglobulin library (e.g., an antibody phage display library) with VTbinding domain to thereby isolate immunoglobulin library members thatbind VT binding domain. Kits for generating, and screening phage displaylibraries are commercially available (e.g., the Pharmacia RecombinantPhage Antibody System, Catalog No. 27-9400-01; and the StratageneSurfZAP™ Phage Display Kit, Catalog No. 240612). Additionally, examplesof methods and reagents particularly amenable for use in generating andscreening antibody display library can be found in, for example, Ladneret al. U.S. Pat. No. 5,223,409; Kang et al. PCT InternationalPublication No. WO 92/18619; Dower et al. PCT International PublicationNo. WO 91/17271; Winter et al. PCT International Publication WO92/20791; Markland et al. PCT International Publication No. WO 92/15679;Breitling et al. PCT International Publication WO 93/01288; McCaffertyet al. PCT International Publication No. WO 92/01047; Garrard et al. PCTInternational Publication No. WO 92/09690; Ladner et al. PCTInternational Publication No. WO 90/02809; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol. Biol.226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram et al.(1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc. Acid Res. 19:4133-4137;Barbas et al. (1991) PNAS 88:7978-7982; and McCafferty et al. Nature(1990) 348:552-554.

[0052] Additionally, recombinant anti-VT binding domain antibodies, suchas chimeric and humanized monoclonal antibodies, comprising both humanand non-human portions, which can be made using standard recombinant DNAtechniques, are within the scope of the invention. Such chimeric andhumanized monoclonal antibodies can be produced by recombinant DNAtechniques known in the art, for example using methods described inRobinson et al. International Application No. PCT/US86/02269; Akira, etal. European Patent Application 184,187; Taniguchi, M., European PatentApplication 171,496; Morrison et al. European Patent Application173,494; Neuberger et al. PCT International Publication No. WO 86/01533;Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European PatentApplication 125,023; Better et al. (1988) Science 240:1041-1043; Liu etal. (1987) PNAS 84:3439-3443; Liu et al. (1987) J. Immunol.139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al.(1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449;and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S.L. (1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214;Winter U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525;Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J.Immunol. 141:4053-4060.

[0053] An anti-VT binding domain antibody (e.g., monoclonal antibody)can be used to isolate VT binding domain by standard techniques, such asaffinity chromatography or immunoprecipitation. An anti-VT bindingdomain antibody can facilitate the purification of natural VT bindingdomain from cells and of recombinantly produced VT binding domainexpressed in host cells. Moreover, an anti-VT binding domain antibodycan be used to detect VT binding domain protein (e.g., in a cellularlysate or cell supernatant) in order to evaluate the abundance andpattern of expression of the VT binding domain protein. Anti-VT bindingdomain antibodies can be used diagnostically to monitor protein levelsin tissue as part of a clinical testing procedure, e.g., to, forexample, determine the efficacy of a given treatment regimen. Detectioncan be facilitated by coupling (i.e., physically linking) the antibodyto a detectable substance. Examples of detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

[0054] Recombinant Expression Vectors and Host Cells

[0055] Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a fusion proteinof the invention, e.g., a nucleic acid sequence encoding a VT bindingdomain (or a portion thereof) operatively linked to at least one othernucleic acid sequence which encodes a polypeptide (e.g., a second domainof a hybrid compound of the invention), in a form suitable forexpression of the fusion protein in a host cell. The term “in a formsuitable for expression of the fusion protein in a host cell” isintended to mean that the recombinant expression vector includes one ormore regulatory sequences operatively linked to the nucleic acidencoding the fusion protein in a manner which allows for transcriptionof the nucleic acid into mRNA and translation of the mRNA into thefusion protein. The term “regulatory sequence” is art-recognized andintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences areknown to those skilled in the art and are described in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). It should be understood that the design of theexpression vector may depend on such factors as the choice of the hostcell to be transfected and/or the amount of fusion protein to beexpressed.

[0056] As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. For example,replication defective retroviruses, adenoviruses and adeno-associatedviruses can be used. Protocols for producing recombinant retrovirusesand for infecting cells in vitro or in vivo with such viruses can befound in Current Protocols in Molecular Biology, Ausubel, F. M. et al.(eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 andother standard laboratory manuals. Examples of suitable retrovirusesinclude pLJ, pZIP, pWE and pEM which are well known to those skilled inthe art. Examples of suitable packaging virus lines include ψCrip, ψCre,ψ2 and ψAm. The genome of adenovirus can be manipulated such that itencodes and expresses a transactivator fusion protein but is inactivatedin terms of its ability to replicate in a normal lytic viral life cycle.See for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld etal. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell68:143-155. Suitable adenoviral vectors derived from the adenovirusstrain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3,Ad7 etc.) are well known to those skilled in the art. Alternatively, anadeno-associated virus vector such as that described in Tratschin et al.(1985) Mol. Cell. Biol. 5:3251-3260 can be used to express a fusionprotein of the invention.

[0057] Certain vectors are capable of autonomous replication in a hostcell into which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

[0058] The recombinant expression vectors of the invention comprise anucleic acid of the invention in a form suitable for expression of thenucleic acid in a host cell, which means that the recombinant expressionvectors include one or more regulatory sequences, selected on the basisof the host cells to be used for expression, which is operatively linkedto the nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory sequence(s)in a manner which allows for expression of the nucleotide sequence(e.g., in an in vitro transcription/translation system or in a host cellwhen the vector is introduced into the host cell). The term “regulatorysequence” is intended to includes promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcell and those which direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences). Itwill be appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, etc.The expression vectors of the invention can be introduced into hostcells to thereby produce proteins or peptides, including fusion proteinsor peptides, encoded by nucleic acids as described herein (e.g., VTbinding domain proteins, mutant forms of VT binding domain, fusionproteins, etc.).

[0059] The recombinant expression vectors of the invention can bedesigned for expression of VT binding domain operatively linked to othermolecules in prokaryotic or eukaryotic cells. For example, VT bindingdomain operatively linked to another molecule can be expressed inbacterial cells such as E. coli, insect cells (using baculovirusexpression vectors) yeast cells or mammalian cells. Suitable host cellsare discussed further in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively,the recombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

[0060] Expression of proteins in prokaryotes is most often carried outin E. coli with vectors containing constitutive or inducible promotorsdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

[0061] Examples of suitable inducible non-fusion E. coli expressionvectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 60-89). Target gene expressionfrom the pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident λ prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter.

[0062] One strategy to maximize recombinant protein expression in E.coli is to express the protein in a host bacteria with an impairedcapacity to proteolytically cleave the recombinant protein (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Another strategy is to alterthe nucleic acid sequence of the nucleic acid to be inserted into anexpression vector so that the individual codons for each amino acid arethose preferentially utilized in E. coli (Wada et al., (1992) NucleicAcids Res. 20:2111-2118). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.

[0063] In another embodiment, the Fusion protein expression vector is ayeast expression vector. Examples of vectors for expression in yeast S.cerivisae include pYepSec1 (Baldari, et al., (1987) Embo J. 6:229-234),pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultzetal., (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego,Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).

[0064] Alternatively, VT binding domain operatively linked to othermolecules can be expressed in insect cells using baculovirus expressionvectors. Baculovirus vectors available for expression of proteins incultured insect cells (e.g., Sf 9 cells) include the pAc series (Smithet al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklowand Summers (1989) Virology 170:31-39).

[0065] In yet another embodiment, a nucleic acid of the invention isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987)Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

[0066] In another embodiment, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid).Tissue-specific regulatory elements are known in the art. Non-limitingexamples of suitable tissue-specific promoters include the albuminpromoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277),lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol.43:235-275), in particular promoters of T cell receptors (Winoto andBaltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al.(1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748),neuron-specific promoters (e.g., the neurofilament promoter; Byrne andRuddle (1989) PNAS 86:5473-5477), pancreas-specific promoters (Edlund etal. (1985) Science 230:912-916), and mammary gland-specific promoters(e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and EuropeanApplication Publication No. 264,166). Developmentally-regulatedpromoters are also encompassed, for example the murine hox promoters(Kessel and Gruss (1990) Science 249:374-379) and the a-fetoproteinpromoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

[0067] The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to VT binding domain mRNA. Regulatory sequencesoperatively linked to a nucleic acid cloned in the antisense orientationcan be chosen which direct the continuous expression of the antisenseRNA molecule in a variety of cell types, for instance viral promotersand/or enhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal., Antisense RNA as a molecular tool for genetic analysis,Reviews—Trends in Genetics, Vol. 1(1) 1986.

[0068] Another aspect of the invention pertains to host cells into whicha recombinant expression vector of the invention has been introduced.The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

[0069] A host cell can be any prokaryotic or eukaryotic cell. Forexample, a fusion protein of the invention can be expressed in bacterialcells such as E. coli, insect cells, yeast or mammalian cells (such asChinese hamster ovary cells (CHO) or COS cells). Other suitable hostcells are known to those skilled in the art.

[0070] Vector DNA can be introduced into prokaryotic or eukaryotic cellsvia conventional transformation or transfection techniques. As usedherein, the terms “transformation” and “transfection” are intended torefer to a variety of art-recognized techniques for introducing foreignnucleic acid (e.g., DNA) into a host cell, including calcium phosphateor calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, or electroporation. Suitable methods fortransforming or transfecting host cells can be found in Sambrook, et al.(Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989), and other laboratory manuals.

[0071] For stable transfection of mammalian cells, it is known that,depending upon the expression vector and transfection technique used,only a small fraction of cells may integrate the foreign DNA into theirgenome. In order to identify and select these integrants, a gene thatencodes a selectable marker (e.g., resistance to antibiotics) isgenerally introduced into the host cells along with the gene ofinterest. Preferred selectable markers include those which conferresistance to drugs, such as G418, hygromycin and methotrexate. Nucleicacid encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding A fusion protein of the invention orcan be introduced on a separate vector. Cells stably transfected withthe introduced nucleic acid can be identified by drug selection (e.g.,cells that have incorporated the selectable marker gene will survive,while the other cells die).

[0072] A host cell of the invention, such as a prokaryotic or eukaryotichost cell in culture, can be used to produce (i.e., express) VT bindingdomain protein operatively linked to other molecules. Accordingly, theinvention further provides methods for producing VT binding domainprotein operatively linked to other molecules using the host cells ofthe invention. In one embodiment, the method comprises culturing thehost cell of invention (into which a recombinant expression vectorencoding VT binding domain operatively linked to other molecules hasbeen introduced) in a suitable medium such that VT binding domainprotein operatively linked to other molecules is produced. In anotherembodiment, the method further comprises isolating VT binding domainoperatively linked to other molecules from the medium or the host cell.

[0073] The host cells of the invention can also be used to producenonhuman transgenic animals. For example, in one embodiment, a host cellof the invention is a fertilized oocyte or an embryonic stem cell intowhich VT binding domain-coding sequences operatively linked to othercoding sequences have been introduced. As used herein, a “transgenicanimal” is a non-human animal, preferably a mammal, more preferably arodent such as a rat or mouse, in which one or more of the cells of theanimal includes a transgene. Other examples of transgenic animalsinclude non-human primates, sheep, dogs, cows, goats, chickens,amphibians, etc. A transgene is exogenous DNA which is integrated intothe genome of a cell from which a transgenic animal develops and whichremains in the genome of the mature animal, thereby directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic animal.

[0074] A transgenic animal of the invention can be created byintroducing VT binding domain-encoding nucleic acid operatively linkedto another nucleic acid molecule into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. Atissue-specific regulatory sequence(s) can be operably linked to the VTbinding domain transgene to direct expression of VT binding domainhybrid protein to particular cells. Methods for generating transgenicanimals via embryo manipulation and microinjection, particularly animalssuch as mice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder etal., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986). Similar methods are used for productionof other transgenic animals. A transgenic founder animal can beidentified based upon the presence of the A fusion protein of theinvention transgene in its genome and/or expression of A fusion proteinof the invention mRNA in tissues or cells of the animals. A transgenicfounder animal can then be used to breed additional animals carrying thetransgene. Moreover, transgenic animals carrying a transgene encoding Afusion protein of the invention can further be bred to other transgenicanimals carrying other transgenes.

[0075] In another embodiment, transgenic non-humans animals can beproduced which contain selected systems which allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. (1992) PNAS89:6232-6236. Another example of a recombinase system is the FLPrecombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991)Science 251:1351-1355. If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0076] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.(1997) Nature 385:810-813. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter Go phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyte and then transferred to pseudopregnant femalefoster animal. The offspring borne of this female foster animal will bea clone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0077] Pharmaceutical Compositions

[0078] The nucleic acid molecules of the invention (e.g., nucleic acidswhich encode a fusion protein of the invention), hybrid compounds of theinvention, and anti-VT binding domain antibodies (also referred toherein as “active compounds”) of the invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically comprise the nucleic acid molecule, hybridcompound, or antibody, and a pharmaceutically acceptable carrier. Asused herein the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, use thereof in thecompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions.

[0079] A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

[0080] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0081] Sterile injectable solutions can be prepared by incorporating theactive compound (e.g., a hybrid compound o the invention) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

[0082] Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0083] For administration by inhalation, the compounds are delivered inthe form of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

[0084] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0085] The compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

[0086] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0087] It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

[0088] The nucleic acid molecules of the invention can be inserted intovectors and used as gene therapy vectors, e.g., as describedhereinabove. Gene therapy vectors can be delivered to a subject by, forexample, intravenous injection, local administration (see U.S. Pat. No.5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994)PNAS 91:3054-3057). The pharmaceutical preparation of the gene therapyvector can include the gene therapy vector in an acceptable diluent, orcan comprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

[0089] The pharmaceutical Compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0090] Uses and Methods of the Invention

[0091] The nucleic acid molecules, hybrid compounds, and antibodiesdescribed herein can be used for methods of treatment. As describedherein, a hybrid compound of the invention can have the followingactivities: (i) it can interact with (e.g., bind to) specific receptorson the surface of a cell, e.g., Gb₃; (ii) while bound to its receptor,it is internalized into the cell; and (iii) it is delivered to aspecific location within the cell. Thus, a VT-binding domain bound(e.g., through a covalent bond) to another moiety can be used to (i)internalize small molecules, e.g., peptides, nucleic acid molecules;(ii) internalize pharmaceutical compositions; and (iii) specificallytarget intracellular locations.

[0092] Thus, in a broad aspect, the invention relates to methods formodulating a cell-associated activity (e.g., cell growth, replication,expression of an endogenous or exogenous gene product, and the like). Anexemplary method includes contacting a cell with a hybrid compound ofthe invention, such that a cell associated activity is altered relativeto the cell-associated activity of the cell in the absence of the hybridcompound.

[0093] The invention also relates to methods for targeting a moiety forinternalization into a Gb₃-containing cell. The method includes the stepof contacting the cell with a hybrid compound of the invention, whereinthe hybrid compound includes the moiety for internalization, such thatthe hybrid compound is internalized into the cell. The moiety can be anymoiety which is to be delivered into the cell, e.g., a toxin (e.g., forkilling the cell), a polynucleotide, e.g., a gene, (e.g., for geneticmodification, e.g., for expression of a gene product in the cell), aprotein or peptide (e.g., an antibody or antigen), and the like. TheGb₃-binding moiety can be, e.g., an anti-Gb₃ antibody, as describedsupra. The targeted moiety can be bound or conjugated to the Gb₃-bindingmoiety as described above.

[0094] Accordingly, the invention relates to methods for treatment,prophylaxis, or diagnosis of a subject, e.g., an animal, including amammal (including both non-human animals and humans). In one embodiment,the invention provides use of a hybrid compound for treatment, therapyor diagnosis of a subject. In one embodiment, the invention provides useof a hybrid compound of the invention for the manufacture of amedicament for the treatment, diagnosis or prophylaxis in a subject. Inone embodiment, a method comprises administering to a subject a hybridcompound of the invention, such that a disease state is treated. Forexample, in one embodiment, the hybrid compound comprises a first domaincomprising VT-B, and a second domian, covalently bound to the firstdomain, comprising a nucleic acid which encodes a gene. Administrationof the hybrid compound (optionally in a pharmaceutically acceptablecarrier) provides a means for delivering the gene to cells which expressGb₃. The cell can then be integrated into the cellular genome and thegene product can be expressed by the cell. For example, the gene couldencode the enzyme adenosine deaminase (ADA), an enzyme which is absentin subjects suffering from ADA deficiency, e.g., as a result of aninborn genetic deficiency. The invention thus relates to methods for thetreatment of genetic defects, e.g., by gene therapy. In anotherembodiment, the hybrid compound comprises a first domain comprisingVT-B, and a second domian, covalently bound to the first domain,comprising an enzyme., e.g., an enzyme which is absent (or present ininsufficient amount) in a subject, or in a tissue of a subject. Forexample, the enzyme could be, e.g., ADA.

[0095] This invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patent applications, patents, and published patentapplications cited throughout this application are hereby incorporatedby reference.

EXAMPLES

[0096] The following materials and methods were used in the Examples:

[0097] Verotoxin Purification

[0098] Recombinant VT binding domain1 from pJLB28 (23), and VT bindingdomain1 B subunit (24) were purified by a recently developed affinitychromatographic technique (25), aliquoted in PBS and stored at −70° C.Purified B subunit was labeled with fluorescein (or rhodamine)isothiocyanate as described (11).

[0099] Cell Culture

[0100] Permanent human malignant astrocytoma cell lines (SF-126, SF-188,SF-539, U 87-MG, U 251-MG, and XF-498 (provided by Dr. J Rutka) HSC(26-29). All cell lines were grown in monolayers in a-MEM (GIBCO) plusnonessential amino acids, glutamine, gentamycin, and 10%heat-inactivated fetal bovine serum, except XF-498 (which was grown inRPMI media) and SKVLB (grown in the presence of 1 μg/ml vinblastine)which is multi-drug resistant variant of the parental SKOV3 ovariancarcinoma cell line (30).

[0101] Verotoxin Cytotoxicity

[0102] Semiconfluent cells in microtiter plates were incubated intriplicates with ten fold VT binding domain dilutions and remainingcells after 72 hr were quantitated by staining with 0.1% crystal violetand measuring the optical density at 570 nm using a microtiter platereader (31).

[0103]FIG. 1 shows the cytotoxic response of six astrocytoma cell linesto increasing concentrations of VT binding domain1. Although each cellline was sensitive to VT binding domain1, a >5000 fold difference insensitivity between the most (SF-539) and least (XF-498) sensitive cellline was apparent.

[0104] We previously showed that the multiple drug resistant ovariancarinoma variant cell lines SKVLB and SKOVC were ˜1000 fold moresensitive to VT binding domain compared to the parental SKOV3 cell line(22).

[0105] Glycolipid Extract of Cultured Cells

[0106] After trypsinization, cells (˜1×10⁶) were washed with PBS threetimes, resuspended in a minimum volume, and extracted with 20 volumes ofchloroform/methanol (C/M) 2:1 by vol. The extract was partitionedagainst water and the lower phase partitioned again against theoreticalupper phase. The combined lower phase was then evaporated, saponifiedwith 1 N NaOH in methanol and glycolipids reextracted as above. Thedried lower phase was dissolved in CM 98:2 and separated by silicachromatography (32). The column was washed extensively with chloroformand glycolipid eluted in acetone/methanol (9:1 by vol.). Gb3 present wasdetected by tlc overlay binding with VT binding domain1 (16).

[0107] Assay of Gb₃ Content by VT Binding Domain1 TLC Overlay

[0108] Aliquots of the glycolipid extract of human tumor samples or oftumor cell lines were separated by TLC {chloroform, methanol,water-65:25:4 (v/v/v)}. The plates were dried and blocked with 1%gelatin in water at 37° C. overnight. They were then washed three timeswith 50 mM TBS for 5 min and incubated with 0.1 g/ml toxin for 1 hour atR.T. After further washing with TBS, plates were incubated with mousemonoclonal anti-VT binding domain1 antibody (33) (2 μg/ml) for VTbinding domain1 followed by peroxidase-conjugated goat anti-mouseantibody. Finally, the plates were washed with TBS, and toxin bindingwas visualized with 4-chloro-1-naphthol peroxidase substrate. A similarplate was prepared and sprayed with orcinol for comparison of glycolipidcontent.

[0109] Since the presence of the toxin receptor glycolipid, Gb₃ isessential to confer sensitivity to VT binding domain, the Gb₃ content ofthe six astrocytoma cell lines analyzed by tlc overlay. Each of theastrocytoma cell lines expressed significant levels of Gb₃. SF-539, themost, and XF-498, the least sensitive cell lines, expressed the highestreceptor level. Thus Gb₃ content is not sufficient to explain the markeddifference in sensitivity to VT binding domain of these cells.

[0110] Previous VT binding domain1 tlc overlay analysis of Gb₃ contentwas also suggestive of increased levels of a slower migrating Gb₃species in SKVLB (22). Comparative HPLC analysis of the fatty acidmethyl esters of SKOV₃ and SKVLB Gb₃ was performed (table 1). As for theastrocytoma cells, no fatty acids shorter than C12 and no hydroxy fattyacids were detected. The results show a marked elevation in short chainfatty acids in SKVLB as compared to SKOV₃ Gb₃, -C16:0 and particularlyC18:0 fatty acids, whereas the content of long chain fatty acids, C22:0,24:0, and 24:1, is greatly reduced in comparison to SKOV3 Gb₃. Thus thechange in Gb₃ fatty acid content for the MDR cell line SKVLB, is similarto the difference observed for SF 529 vs XF 498 cells and the changefound following butyrate treatment of XF 498 cells. In each case,increased sensitivity to VT binding domain1 correlated with an increasedproportion of short chain fatty acid Gb₃ species.

[0111] Cell Sensitization to VT Binding Domain by Butyrate Treatment

[0112] XF-498 astrocytoma, ovarian tumour and vero cell lines werecultured in media containing 2 mM sodium butyrate (or propionate orcapronate for astrocytoma cells).

[0113] Sodium butyrate has been found to increase sensitivity to VTbinding domain in several cell systems (12, 13, 43, 44). We thereforedetermined the effect of butyrate on the VT binding domain sensitivityof XF-498 cells.

[0114] morphology and growth: Initial studies showed butyrate treatmentof XF-498 cells had a profound effect on the morphology of these cells.XF-498 cells are small, round cells that pile up, and even uponconfluency, do not form a monolayer. In contrast, SF-539 cells are flat,stellate and form a confluent monolayer. Butyrate treated XF-498 cellsadopt a similar morphology to SF-539 cells. XF498 cells cultured inpropionate showed a similar, but less significant, morphological changebut culture of XF498 cells in capronate had no effect. PPMP inhibitionof glycolipid biosynthesis (45) prevented the butyrate-inducedmorphological changes. PPMP alone had no effect on XF 498 morphology.

[0115] VT binding domain1 sensitivity: In addition to effecting amorphological change to more resemble SF596 cells, butyrate induced asignificant (5000 fold) increase in XF498 cell sensitivity to VT bindingdomain1. VT binding domain1 sensitivity was increased to a lesser extentfor propionate treated cells, but capronate had no effect. PPMPprevented butyrate induced VT binding domain1 sensitivity of XF-498cells (not shown) as Sandvig has reported (46).

[0116] subcellular VT binding domain targeting: Concommittant with theincreased sensitivity to VT binding domain1 induced by butyratetreatment of XF 498 cells, butyrate induced a marked change in theintracellular routing of VT binding domain1 B, such that the toxinbecame localized mainly around the ER/nuclear membrane as for SF-539.Serial sectioning shows the B subunit to be, in part, located within arestricted region of the nucleus. Pixel integration of “z” scans acrossthe nucleus in the composite confocal images shows three labeling maximacorresponding to the nuclear boundaries and a central intranuclear peakin SF 529 cells but a single juxtanuclear peak in XF 498 cells withoutperi- or intranuclear staining. After butyrate treatment, the majorlabeling for XF 498 cells is within the nucleus.

[0117] Immunoelectron microscopy further confirmed the nuclear locationof VT binding domain1 B in SF-539 and butyrate treated XF-498 cells. Inuntreated XF 498 cells, VT binding domain1B was detected in the Golgibut nuclear labeling was not above background.

[0118] Gb₃ expression: Butyrate treatment has been previously found toinduce Gb₃ synthesis to mediate increased VT binding domain sensitivity(43, 46). The Gb₃ content of SF 539 cell was therefore compared to thatof XF 498 cells before and after butyrate treatment by VT bindingdomain1-tlc overlay. It can be seen that the Gb₃ contained within XF 498cells runs on tlc as single band corresponding to the faster Gb₃ band ofthe renal standard. In contrast, SF 539 cells contain an additionalslower migrating Gb₃ band. Following butyrate treatment, there is adramatic, selective increase in this slower Gb₃ band in XF 498 cells,such that this band now becomes the dominant VT binding domain1 bindingGb₃ species. This Gb₃ species is also selectively increased, but to alesser extent, following propionate treatment, whereas capronate has noeffect.

[0119] Gb₃ fatty acid isoform expression: HPLC analysis of the fattyacid composition of the Gb₃ purified from SF 539 and XF 498 cells beforeand after butyrate treatment showed that the SF 539 Gb₃ species wasenriched in short chain fatty acids (C16 and C18) and deficient in thelonger chain species (C22, C24) relative to XF 498 Gb₃. Subfractionation of the Gb₃ species in butyrate treated XF 498 into slow,‘intermediate’ and fast fractions confirmed the markedly increased C16and decreased C24 fatty acid content of the slow migrating Gb₃ speciesinduced by butyrate.

[0120] Cell surface VT binding domain1 binding: To preclude thedifferential VT binding domain1 sensitivity of astrocytoma cells beingdue to differences in toxin binding, cell surface binding at 4° C. wasquantitated using ¹²⁵I-labeled VT binding domain1. The comparison ofXF498 (with and without butyrate treatment) and SF 539 is shown in FIG.2. All three cell ‘types’ demonstrate comparable VT binding domain1binding. If anything, the more VT binding domain susceptible butyratetreated cells show less VT binding domain binding.

[0121] Electron Microscopy

[0122] Targeting of VT binding domain1 in SF-539 and XF-498 cells wasexamined in detail by transmission EM. Cells were cultured on atransferable membrane inserts in 24 well tissue culture plates andallowed to form a confluent monolayer. For surface binding, the cellswere incubated at 4° C. in the presence of 5 μg/ml VT1B for 30 min andwashed with cold PBS. Then, cells were incubated with anti-VT1B primaryantibody followed by GAM-gold secondary antibody for 30 min at 4° C. Fortoxin internalization studies, cells were incubated with VT 1 B (5μg/ml) at 37° C. for 1 hr. For either treatment, the cells were fixed in2.5% gluteraldehyde, 2% paraformaldehyde in PBS for 30 min at roomtemperature. After washing with PBS, cells were post fixed in 1% osmiumtetroxide in phosphate buffer for 30 min and washed with saline. Thecells were further postfixed with 2% uranyl acetate in 30% ethanol for15 min (38), and dehydrated in an ethanol gradient. Dehydrated cellswere serially infiltrated with 50% to 75% Epon in 100% ethanol for 30min each and finally with 100% Epon for 1 h with 2 changes of Epon. Themembrane was removed from the holder and inserted vertically into thegelatin capsule, filled with 100% Epon and polymerized at 65° C.

[0123] After sectioning of the polymerized block on an ultramicrotome,the sections for surface binding studies were counter stained, while thesections for studies of internalization were immunolabeled. Thesesections were treated with saturated sodium metaperiodate for 30 min tounmask antigenicity (39) and washed in distilled water. The sectionswere next blocked by floating on a drop of 0.1% BSA, 0.2% fish skingelatin in 50 mM TBS (150 mM NaCl, 50 mM Tris, pH 7.4) for 30 min andimmunolabeled with 1:50 dilution of anti-VT binding domain1B antibody,followed by 1:50 dilution of GAM-15 or -10 nm gold for 1 h each at roomtemperature, with through washing after each step. Finally, the sectionswere stained with 5% uranyl acetate and then with Reynold's lead citratefor 6 min each and analyzed under a Philips 300 EM microscope at 60 kV.

Example 1 Preparation of a Labeled Hybrid VT Binding Domain Moiety

[0124] Lysosomal Labeling (FITC-Dextran and RITC-VT Binding Domain1BDouble Labeling)

[0125] Internalization of FITC-dextran was used as a lysosomal marker aspreviously (34). Cells grown overnight on cover slips were incubatedwith 0.5 μg/ml FITC-dextran in a-MEM for 24 hrs at 37° C. and werechased with fresh medium for 2 hrs at the same temperature. RITC-VTbinding domain1 B (2-5 μg/ml) was added to the cells in last 1 hr ofchase at 37° C. The cells were then washed five times with sterile PBS,fixed in 2% formaldehyde, mounted with DABCO (35) and visualized underincident fluorescent light using a Polyvar fluorescent microscope.Fluorescent images were recorded on Kodak TMAX 400 ASA film.

[0126]¹²⁵I-Verotoxin Binding of Intact Cells

[0127] Cells were grown on 96 well plates. Media was aspirated, and thecells were washed with PBS (pH 7.4) at room temperature. Afterequilibrating the cells on ice, 100 μL of ice-cold PBS was added to eachwell. The radiolabeled toxin was added and allowed to incubate for onehour on ice. Unbound toxin was then washed off with ice cold PBS 3times. The cells were solublized with 1 ml of 10% SDS added to each welland incubated at 37° C. for 15 minutes and the extracts counted in agamma counter.

[0128] Inhibition of Glycolipid Synthesis

[0129] PPMP(1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol,Matreya Inc., Pleasant Gap, Pa.) is a potent inhibitor ofglucosylceramide synthesis (36) and thereby prevents the synthesis ofmost glycolipids. Experiments extablished that 27 μM PPMP was able toinhibit glycosphingolipid synthesis without inducing cell death. XF-498astrocytoma and vero cells were then incubated at 37° C. with 27 μM PPMPin the presence and absence of 2 mM butyrate for 6-7 days.

[0130] Fluorescence Microscopy

[0131] FITC-VT binding domain1 B was prepared as described before (11,37). Briefly 50 μg of purified VT binding domain1 was dialyzed againstborate buffer (50 mM, pH 9.0) overnight. Fluorescein isothiocyanate(FITC,) (10 mg/ml) was added to the dialyzed VT binding domain1B-subunitand allowed to react in the dark for 2 hours at 4° C. and then dialyzedagainst PBS. The fluorescein-conjugated VT binding domain1B was storedat −70° C. Rhodamine conjugated VT binding domain1B (RITC-VT bindingdomain1B) was similarly prepared. To determine the subcellular targetingof VT binding domain1 in astrocytoma cells, SF-539 and XF-498 cellsgrown overnight on glass cover slips were incubated for 1 hr in thepresence of 10 μg/ml of FITC-VT binding domain1 B at 37° C. for VTbinding domain1 internalization. Following extensive washing with 50 mMPBS, cell were mounted with DABCO and examined under the fluorescencemicroscope.

[0132] Confocal Microscopy

[0133] To determine the three dimensional topology of VT binding domain1subcellular targeting, tumor cells grown on coverslips were incubated inthe presence of 10 μg/ml FITC-VT binding domain1B at 37° C. for 1 hr.Following extensive washing with 50 mM PBS, the cells were mounted withDABCO and examined by confocal microscopy using a laser confocalmicroscope with 40× objective. For double labeling RITC-VT bindingdomain1B-labeled cells were fixed, permeablized with saposin or 0.1%Triton and treated with rabbit anti ERGIC 53 (kindly provided by Dr H.Hauri, Biozentrum, Basel) or anti BIP (directed against amino acids andnot cross reactive with other hsp70s, Santa Cruz Ltd) followed by FITClabeled rabbit antimouse Ig, or FITC-labeled ConA, and washedextensively. For lysosomal double labeling, cells were pretreated withFITC labeled dextran overnight prior to incubation with RITC-VT bindingdomain1B for 1 hr.

[0134] A krypton/argon laser tuned to produce both 488 nm and 565 nmwavelength beams, was used for fluorescein and rhodamine excitation,respectively. The dual filter block, K1K2 and allowed the simultaneousmonitoring of the fluorescein and rhodamine emission. The signals wererecorded by optical sections of 1 μm thickness. Diaphragm andfluorescence detection level were adjusted in order to avoid anycross-emission from two channel (FITC and RITC). Pictures were recordedwith a Kalman filter (average of six images) and transferred to KodakT-max 400 film. Computational analysis of the digital image allowedprecise identification of double labeled structures.

Example 2 RME of a Labeled Hybrid VT Binding Domain Moiety

[0135] Subcellular Targeting of VT Binding Domain1B in Astrocytoma CellLines

[0136] The intracellular routing of VT binding domain1, following RME,was monitored in SF-539 and XF-498 cells, using FITC-labeled B subunit.Similar results were obtained using FITC-VT binding domain1 (data notshown).

[0137] The pattern of FITC-VT binding domain1 B localization wascompletely distinct in the SF-539 and XF-498 astrocytoma cell lines,despite comparable Gb3 content and cell surface binding at 4° C. In themore VT binding domain sensitive SF-539 cells, at 37° C., intracellularFITC-VT binding domain1-B accumulated around the nucleus and apparentlywithin the nucleus. However, the intracellular localization of FITC-VTbinding domain1-B in XF-498 cells was in a juxtanuclear location,consistent with Golgi localization. Double labeling confocal microscopyverified the targeting of VT binding domain1B to the the nuclearenvelope/ER and nucleus in SF-539 cells. In SF-539 cells, RITC-B alsocolocalized with anti BIP (GRP 78), a marker for the ER(41) as a ringaround the nucleus. The punctate staining for VT binding domain1B andBIP for the most part was coincident, however some BIP staining showedno corresponding toxin localization and vice versa. The latter result islikely due to VT binding domain1B localization in part, in intermediatecompartment vesicles (between Golgi and ER) (42). In addition,intranuclear staining is clearly seen for VT binding domain1B but notfor BIP. Staining for ERGIC 53, a marker of the intermediate compartmentvesicles in part, colocalized with VT binding domain1B staining. Incontrast, no nuclear staining was seen for XF-498. Double labelingconfocal microscopy showed that the juxtanuclear structure labeled in XF498 cells was colocalized with Con A labeled Golgi. VT binding domain1Bwas restricted to the Golgi and did not localize with the additional ConA. staining of the ER around the nucleus.

[0138] A degree of colocalization of RITC-labeled VT binding domain1Bwith FITC-dextran (lysosomal marker) was observed in XF-498 cellssuggesting that the toxin is internalized through lysosomal/endosomalvesicles in these cells. In SF-539 cells (and butyrate-treated XF-498cells-not shown) FITC-dextran was not colocalized with any RITC-VTbinding domain1B.

[0139] Confocal microscopy shows that FITC-VT binding domain1-B wasdifferentially targeted in SKVLB and SKOV3 cells. In SKVLB, cells theinternalized toxin is found within the nuclear membrane/ER and nucleus,whereas in SKOV3 cells, the majority of intracellular toxin is in ajuxtanuclear location, consistent with Golgi targeting. Thus,intracellular VT binding domain1B is localized in SKVLB in a mannersimilar to SF 529 and butyrate treated XF 498 cells, whereas in SKOV3cells, toxin is localized as in XF 498 cells.

Example 3 Preparation of VT Binding Domain Fusion Protein

[0140] Recombinant VT binding domain can be produced in a variety ofexpression systems. For example, the mature VT binding domain peptidecan be expressed as a recombinant glutathione-S-transferase (GST) fusionprotein in E. coli and the fusion protein can be isolated andcharacterized. Specifically, as described above, VT binding domain(e.g., VT-B) can be fused to GST and this fusion protein can beexpressed in E. coli strain PEB 199. As GST is predicted to be 26 kD,the fusion protein is predicted to be about 26 kD in molecular weightgreater than VT-B. Expression of the GST-VT binding domain fusionprotein in PEB 199 can be induced with IPTG. The recombinant fusionprotein can be purified from crude bacterial lysates of the induced PEB199 strain by affinity chromatography on glutathione beads.Polyacrylamide gel electrophoretic analysis of the proteins purifiedfrom the bacterial lysates results in isolation of the fusion protein asa band about 26 kD in molecular weight greater than VT-B.

[0141] The preparation and purification of modified VT binding domainhaving a carboxy-terminal tetrapeptide has been reported (Johannes etal. (1997) J. Biol. Chem. 272:19554-19561) by using a pSU108-basedplasmid expressing the VT binding domain. Fusion proteins according tothe invention can be also be purified and labeled as described therein.

Example 4 Preparation of VT Binding Domain DNA Fusion Molecule

[0142] Construction of VT1 B subunit λ Cro Fusion Proteins for Transferof Specific DNA Sequences into the Cell Nucleus.

[0143] λ Cro is a 66 amino acid helix-turn-helix DNA-biding protein frombacteriophage λ. This protein binds as a dimer to a 17 base pair λ.operator sequence and acts as a transcription repressor. A stablemonomeric form of λ. Cro has been designed which exhibits similarstructure to wild-type Cro although with reduced DNA binding capacity(e.g., Mossing and Sauer, Science (1990) 250 (4988):1712-5).

[0144] To construct the VTIB-Cro fusion protein, the complete wild-typeλ. Cro coding sequence from plasmid pUCroRX (gift of M. Mossing,University of Notre Dame, Ind.) is inserted downstream and in frame withthe complete VT1 B sequence in plasmid pJLB120 (e.g., Ramotar, et al.,Biochem. J. (1990) 272 (3): 805-11). pJLB120 is a derivative of thepKK223-3 E. coli expression vector. The stop codon in VT1B is eliminatedand replaced with a short linker sequence coding for 6-12 hydrophilicresidues. This enables both VT1B subunit and Cro domains to exhibitproper folding and molecular interactions. VT1B-Cro is expressed in E.coli, and affinity purified by binding to Gb₃. To reconstitute fusionprotein-DNA binding, the VT1B-Cro fusion protein is incubated withpurified (or in vitro translated) wild-type λ. Cro, then this complex isallowed to bind DNA fragments containing λ. operator sequences derivedfrom plasmid ptacλ.1 (e.g., Mossing et al., Methods Enzymol. (1991) 208:604-19).

[0145] Alternatively, the fusion protein is constructed as describedabove, except the wild-type Cro sequence will be replaced with theengineered Cro monomer sequence from plasmid pUCro.mDG (e.g., Mossingand Sauer, Science (1990) 250 (4988):1712-5). Purified VT1B-fusionprotein is incubated with DNA fragments containing the λ. operator andtested for DNA binding by Electrophoretic Mobility Shift Assay or byDNase I analysis.

[0146] The fusion protein constructed as above can be bound to a DNA forwhich it is selective, and thereby be used to transport the DNA into acell by receptor-mediated endocytosis.

Example 5 Preparation of a VT-B/Polylysine Hybrid Compound

[0147] Commercially-available polylysine (MW1-4000) was activated usingEDC (1-ethyl-3(3-dimethylaminopropyl) carbodiimide in the presence ofN-hydroxysuccinimide, using the method of Grabarek and Gergely (“ZeroLength Crosslinking Procedures with the use of Active Esters” AnalyticalBiochemistry 185:131-134,1990). The resulting succinimidyl ester wasthen reacted with the verotoxin 1 B-subunit (purified as describedsupra), in 0.1 M MES buffer, 0.5M NaCl, pH 6.0, for one hour at roomtemperature. Crosslinking was monitored by Tricine-SDS-PAGE(polyacrylamide gel eletrophoresis). The PAGE analysis showed thepresence of higher molecular weight bands, corresponding to theVT-B/polylysine conjugate.

[0148] The VT-B/polylysine conjugate (hybrid compound) is purified byroutine methods and is then used complex with a DNA. The hybridmolecule/DNA complex can then be administered to a cell (or to a livingsubject) to deliver the DNA to the cell.

REFERENCES

[0149] 1. Sandvig, K., O. Garred, P. K. Holm and B. van Deurs. 1993.Endocytosis and intracellular transport of protein toxins., Biochem SocTransact, 21, 707.

[0150] 2. Olsnes, S., B. van Deurs and K. Sandvig. 1993. Protein toxinsacting on intracellular targets: cellular uptake and translocation tothe cytosol, Med Microbiol Immunol, 182, 51.

[0151] 3. Endo, Y., K. Tsurugi, T. Yutsudo, Y. Takeda, K. Ogasawara andK. Igarashi. 1988. Site of the action of a vero toxin (VT bindingdomain2) from Escherichia coli O157:H7 and a Shiga toxin on eukaryoticribosomes, Eur J Biochem, 171, 45.

[0152] 4. Saxena, S. K., A. D. O'Brien and E. J. Ackerman. 1989. Shigatoxin, Shiga-like toxin II variant, and ricin are all single-site RNAN-glycosidases of 28 S RNA when microinjected into Xenopus oocytes, JBiol Chem, 264, 596.

[0153] 5. Mangeney, M., C. A. Lingwood, B. Caillou, S. Taga, T. Turszand J. Wiels. 1993. Apoptosis induced in Burkitt's lymphoma cells viaGb3/CD77, a glycolipid antigen., Cancer Res, 53, 5314.

[0154] 6. Arab, S., M. Murakami, P. Dirks, B. Boyd, S. Hubbard, C.Lingwood and J. Rutka. submitted. Verotoxins inhibit the growth of andinduce apotosis in human astrocytoma cells.,

[0155] 7. Inward, C.D., J. Williams, I. Chant, J. Crocker, D. V.Milford, P. E. Rose and C. M. Taylor. 1995. Verocytotoxin-1 inducesapoptosis in vero cells, J Infect, 30, 213.

[0156] 8. Griffin, P. M. 1995. Escherichia coli O157:H7 and otherenterohemorrhagic Escherichia coli. In Infections of theGastrointestinal Tract, M. J. Blaser, P. D. Smith, J. I. Ravdin, H. B.Greenberg and R. L. Guerrant, Raven Press, Ltd., New York, 739.

[0157] 9. Rowe, P., E. Orrbine, G. Wells and P. McLaine. 1991.Epidemiology of the hemolytic-uremic syndrome in Canadian children from1986-1988, J Pediat, 119, 218.

[0158] 10. Gannon, V. P. J. and C. L. Gyles. 1990. Characteristics ofthe Shiga-like toxin produced by Escherichia coli associated withporcine edema disease, Vet Microbiol, 89.

[0159] 11. Khine, A. A. and C. A. Lingwood. 1994. Capping and receptormediated endocytosis of cell bound verotoxin(Shiga-like toxin) 1;Chemical identification of an amino acid in the B subunit necessary forefficient receptor glycolipid binding and cellular internalization., JCell Physiol, 161, 319.

[0160] 12. Sandvig, K., K. Prydz, M. Ryd and B. van Deurs. 1991.Endocytosis and intracellular transport of the glycolipid-binding ligandShiga toxin in polarized MDCK cells, J Cell Biol, 113, 553.

[0161] 13. Sandvig, K., M. Ryd, O. Garred, E. Schweda and P. K. Holm.1994. Retrograde transport from the Golgi complex to the ER of bothShiga toxin and the nontoxic Shiga B-fragment is regulated by butyricacid and cAMP, J Cell Biol, 126, 53.

[0162] 14. Sandvig, K. and B. Van Deurs. 1996. Endocytosis,intracellular transport, and cytotroxic action of Shiga toxin andricin., Physiol Rev, 76, 949.

[0163] 15. Pellizzari, A., H. Pang and C. A. Lingwood. 1992. Binding ofverocytotoxin 1 to its receptor is influenced by differences in receptorfatty acid content, Biochem, 31, 1363.

[0164] 16. Kiarash, A., B. Boyd and C. A. Lingwood. 1994.Glycosphingolipid receptor function is modified by fatty acid content:Verotoxin 1 and Verotoxin 2c preferentially recognize differentglobotriaosyl ceramide fatty acid homologues, J Biol Chem, 269, 11138.

[0165] 17. Arab, S. and C. A. Lingwood. 1996. Influence of phospholipidchain length on verotoxin/globotriaosyl ceramide binding in modelmembranes: comparison of a surface bilayer film and liposomes, GlycoconjJ, 13, 159.

[0166] 18. Nyholm, P. -G., G. Magnusson and C. Lingwood. 1996. Twodistinct binding sites for globotriaosyl ceramide on verotoxins:molecular modelling and confirmation by analogue studies and a newglycolipid receptor for all verotoxins, Chem Biol, 3, 263.

[0167] 19. Arab, S., A. Khine, J. Rutka, S. Grinstein and C. Lingwood.1995. Globotriaosyl ceramide-mediated intracellular targetting ofverotoxin. Retrograde transport to the nucleus and beyond, Glycoconj J,24, 499.

[0168] 20. Lingwood, C. A. 1996. Aglycone Modulation of GlycolipidReceptor Function, Glycoconj J, 13, 495.

[0169] 21. Sandvig, K., O. Garred, K. Prydz, J. Kozlov, S. Hansen and B.van Deurs. 1992. Retrograde transport of endocytosed Shiga toxin to theendoplasmic reticulum., Nature, 358, 510.

[0170] 22. Farkas-Himsley, H., B. Rosen, R. Hill, S. Arab and C. A.Lingwood. 1995. Bacterial colicin active against tumour cells in vitroand in vivo is verotoxin 1, Proc Natl Acad Sci, 92, 6996.

[0171] 23. Petric, M., M. A. Karmali, S. Richardson and R. Cheung. 1987.Purification and biological properties of Escherichia coliverocytotoxin, FEMS Microbiol Letts, 41, 63.

[0172] 24. Ramotar, K., B. Boyd, G. Tyrrell, J. Gariepy, C. A. Lingwoodand J. Brunton. 1990. Characterization of Shiga-like toxin 1 B subunitpurified from overproducing clones of the SLT-1 B cistron, Biochem J,272, 805.

[0173] 25. Boulanger, J., M. Huesca, S. Arab and C. A. Lingwood. 1994.Universal method for the facile production of glycolipid/lipid matricesfor the affinity purification of binding ligands, Anal Biochem, 217, 1.

[0174] 26. Rutka, J., J. Giblin, H. Hoifodt, D. Dougherty, C. Bell, J.McCullough, R. Davis, C. Wilson and M. Rosenblum. 1986. Establishmentand characterization of a cell line from a human gliosarcoma, CancerRes, 46, 5893.

[0175] 27. Rutka, J. T., J. R. Giblin, D. Y. Dougherty, H. C. Liu, J. R.McCulloch, C. W. Bell, R. S. Stem, C. B. Wilson and M. L. Rosenblum.1987. Establishment and characterization of five cell lines derived fromhuman malignant gliomas, Acta Neuropathol (Berl), 75, 92.

[0176] 28. Ponten, J. and E. MacIntyre. 1986. Long-term culture ofnormal and neoplastic human glia, Pathol Microbiol Scand, 81, 791.

[0177] 29. Westermark, B., J. Ponten and R. Hugosson. 1973. Determinantsfor the establishment of permanent tissue culture lines from humangliomas, Acta Pathol Microbiol Scand, 81, 791.

[0178] 30. Bradley, G., M. Naik and V. Ling. 1989. P-glycoproteinexpression in multidrug-resistant human ovarian carcinoma cell lines,Canc Res, 49, 2790.

[0179] 31. Kueng, W., E. Silber and U. Eppenberger. 1989. Quantificationof cells cultured on 96-well plates, Anal Biochem, 182, 16.

[0180] 32. Boyd, B. and C. A. Lingwood. 1989. Verotoxin receptorglycolipid in human renal tissue, Nephron, 51, 207.

[0181] 33. Boulanger, J., M. Petric, C. A. Lingwood, H. Law, M. Roscoeand M. Karmali. 1990. Neutralization receptor-based immunoassay(NeutrELISA) for detection of neutralizing antibodies to Escherchia coliverocytotoxin 1, J Clin Micro, 28, 2830.

[0182] 34. Kim, J. H., A. A. Khine, C. A. Lingwood, W. Furuya, M. F.Manolson and S. Grinstein. 1996. Dynamic measurement of the pH of theGolgi complex in living cells using retrograde transport of theverotoxin receptor, J Cell Biol, 134, 1387.

[0183] 35. Krenik, K. D., G. M. Kephart, K. P. Offord, S. L. Dunnetteand G. J. Gleich. 1989. Comparison of antifading agents used inimmunofluorescence, J Immunol Methods, 117,91.

[0184]36. Inokuchi, J. -I. and N. S. Radin. 1987. Preparation of theactive isomer of 1-phenyl-2-decanoylamino-3-morpholino-1-propanol,inhibitor of murine glucocerebroside synthetase, J Lipid Res, 28, 565.

[0185] 37. Lingwood, C. A. 1994. Verotoxin-binding in human renalsections, Nephron, 66, 21.

[0186] 38. Pulczynski, S., A. M. Boesen and O. M. Jensen. 1993.Antibody-induced modulation and intracellular transport of CD10 and CD19antigens in human B-cell lines: An immunofluorescence and immunoelectronmicroscopy study., Blood, 81, 1549.

[0187] 39. Stirling, J. W. and P. S. Graff. 1995. Antigen unmasking forimmunoelectron microscopy: labeling is improved by treating with sodiumethoxide or sodium metaperiodate, then heating on retriveal medium, JHistochem Cytochem, 43, 115.

[0188] 40. Myher, J. J., A. Kuksis and S. Pind. 1989. Molecular speciesof glycerolipids and sphingomyelins of human erythrocytes: improvedmethod of analysis, Lipids, 24, 396.

[0189] 41. Haas, I. G. 1994. BiP (GRP78), an essential hsp70 residentprotein in the endoplasmic reticulum., Experientia, 50, 1012.

[0190] 42. Kappeler, F., C. Itin, R. Schindler and H. P. Hauri. 1994. Adual role for COOH-terminal lysine residues in pre-Golgi retention andendocytosis of ERGIC-53, J Biol Chem, 269, 6279.

[0191] 43. Louise, C. B., S. A. Kaye, B. Boyd, C. A. Lingwood and T. G.Obrig. 1995. Shiga toxin-associated hemolytic uremic syndrome: Effect ofsodium butyrate on sensitivity of human unbilical vein endothelial cellsto Shiga toxin, Infect Immun, 63, 2765.

[0192] 44. Keusch, G. T., D. W. K. Acheson, L. Aaldering, J. Erban andM. S. Jacewicz. 1996. Comparison of the effects of Shiga-like toxin 1cytokine-and butyrate pretreated human umbilical and saphenous veinendothelial cells, J Infect Dis, 173, 1164.

[0193] 45. Abe, A., J. A. Shayman and N. S. Radin. 1996. A novel enzymethat catalyzes the esterification of N-acetylsphingosine, J Biol Chem,271, 14383.

[0194] 46. Sandvig, K., O. Garred, A. van Helvoort, G. van Meer and B.van Deurs. 1996. Importance of glycolipid synthesis for butyricacid-induced sensitization to Shiga toxin and intracellular sorting oftoxin in A431 cells, Mol Biol Cell, 7, 1391.

[0195] 47. Lingwood, C. A., H. Law, S. Richardson, M. Petric, J. L.Brunton, S. DeGrandis and M. Karmali. 1987. Glycolipid binding ofpurified and recombinant Escherichia coli-produced verotoxin in vitro, JBiol Chem, 262, 8834.

[0196] 48. Waddell, T., S. Head, M. Petric, A. Cohen and C. A. Lingwood.1988. Globotriosyl ceramide is specifically recognized by the E. coliverocytotoxin 2, Biochem Biophys Res Commun, 152, 674.

[0197] 49. Boyd, B., Z. Zhiuyan, G. Magnusson and C. A. Lingwood. 1994.Lipid modulation of glycolipid receptor function: Presentation ofgalactose a1-4 galactose disaccharide for Verotoxin binding in naturaland synthetic glycolipids., Eur J Biochem, 223, 873.

[0198] 50. St. Hilaire, P. M., M. K. Boyd and E. J. Toone. 1994.Interaction of the Shiga-like toxin type 1 B-subunit with itscarbohydrate receptor, Biochem, 33, 14452.

[0199] 51. Head, S., M. Karmali and C. A. Lingwood. 1991. Preparation ofVT binding domain1 and VT binding domain2 hybrid toxins from theirpurified dissociated subunits: Evidence for B subunit modulation of Asubunit function, J Biol Chem, 266, 3617.

[0200] 52. Louise, C. B., T. P. Moran, C. A. Lingwood, P. J. DelVecchio, D. J. Culp,. and T. G. Obrig. 1995. “Binding of [¹²⁵I]Shiga-like toxin-i to human endothelial cells: implications for thepathogenesis of Shiga toxin-associated hemolytic uremic syndrome”,Endothelium, 3, z,900 159.

[0201] 53. Robinson, L. A., R. M. Hurley, C. A. Lingwood and D. G.Matsell. 1995. “Escherichia coli verotoxin binding to human paediatricglomerular mesangial cells”, Ped Nephrol, 9, 700.

[0202] 54. Cohen, A., V. Madrid-Marina, Z. Estrov, M. Freedman, C. A.Lingwood and H. -M. Dosch. 1990. Expression of glycolipid receptors toShiga-like toxin on human B lymphocytes: a mechanism for the failure oflong-lived antibody response to dysenteric disease, Int Immunol, 2, 1.

[0203] 55. Jacewicz, M. S., D. W. K. Acheson, M. Mobassaleh, A.Donohue-Rolfe, K. A. Balasubramanian and G. T. Keusch. 1995.Maturational regulation of globotriaosylceramide, the Shiga-like toxin 1receptor, in cultured human gut epithelial cells, J Clin Invest, 96,1328.

[0204] 56. Jacewicz, M., H. A. Feldman, A. Donohue-Rolfe, K. A.Balasubramanian and G. T. Keusch. 1989. Pathogenesis of Shigelladiarrhea. XIV. Analysis of Shiga toxin receptors on cloned HeLa cells.,J Infect Dis, 159, 881.

[0205] 57. Eiklid, K. and S. Olsnes. 1980. Interaction of Shigellacytotoxin with receptors on sensitive and insensitive cells, J ReceptRes, 1, 199.

[0206] 58. Johannes, L., D. Tenza, C. Anthoy and B. Goud. 1997.Retrograde transport of KDEL-bearing B-fragment of Shiga toxin, J BiolChem, 272, 19554.

[0207] 59. Kim, J. H., L. Johannes, B. Goud, C. Antony, C. A. Lingwood,R. Daneman and S. Grinstein. submitted. “Non-invasive measurement of thepH of the endoplasmic reticulum at rest and during calcium release”,

[0208] 60. Maloney, M. D. and C. A. Lingwood. 1994. CD19 has a potentialCD77 (globotriaosyl ceramide)-binding site with sequence similarity toverotoxin B-subunits: Implications of molecular mimicry for B celladhesion and enterohemorrhagic Escherichia coli pathogenesis, J Exp Med,180, 191.

[0209] 61. Khine, A. A. and C. A. Lingwood. submitted. CD77 DependentRetrograde Transport of CD19 to the Nuclear Membrane: FunctionalRelationship between CD77 and CD19 during Germinal Center B-cellApoptosis.,

[0210] 62. Jans, D. 1994. Nuclear signaling pathways for polypeptideligands and their membrane receptors?, FASEB, 8, 841.

[0211] 63. Stachowiak, M., P. Maher, E. Mordechai and E. Stachowiak.1996. Nuclear accumulation of fibroblast growth facto receptors isregulated by multiple signals in adrenal medullary cells., Mol BiolCell, 7, 1299.

[0212] 64. Gillard, B. K., L. T. Thurman, R. G. Harrell, Y. Capentanaki,M. Saito, R. K. Yu and D. M. Marcus. 1994. Biosynthesis ofglycosphingolipids is reduced in the absence of a vimentin; intermediatefilament network, J Cell Science, 107, 3545.

[0213] 65. van Helvoort, A., A. Smith, H. Sprong, I. Fritzsche, A.Schinkel, P. Borst and G. van Meer. 1996. MDR1 P-Glycoprotein is a lipidtranslocase of broad specificity, while MDR3 P-glycoprotein specificallytranslocates phosphatidyl choline., Cell, 87, 507.

[0214] 66. Lavie, Y., H. Cao, S. L. Bursten, A. E. Giuliano and M. C.Cabot. 1996. Accumulation of glucosylceramides in multidrug-resistantcancer cells, J Biol Chem, 271, 19530.

[0215] 67. Burger, K., P. van der Bijl and G. van Meer. 1996. Topologyof sphingolipid galactosyl transferase in ER and Golgi:transbilayermovement of monohexyl sphingolipids is required for higherglycosphingolipid biosynthesis., J Cell Biol, 133, 15.

[0216] 68. Bates, S. E., L. Mickley, Y. Chen, N. Richert, J. Rudick, J.Biedler and A. Fojo. 1989. Expression of a drug resistance gene in humanneuroblastoma cell lines:modulation by retenoic acid-induceddifferentiation., Moll Cell Biol, 9, 4337.

[0217] 69. Bates, S. E., S. J. Currier, M. Alvarez and A. T. Fojo. 1992.Modulation of P-glycoprotein phosphorylation and drug transport bysodium butyrate, Biochem, 31, 6366.

[0218] 70. Arab, S., E. Russel, W. Chapman, B. Rosen and C. Lingwood. inpress. Expression of the Verotoxin receptor glycolipid, globotriaosylceramide in Ovarian Hyperplasias, Oncol Res,

[0219] 71. Newburg, D., P. Chaturvedi, E. Lopez, S. Devoto, A. Feyad andT. Cleary. 1993. Susceptibilty to hemolytic-uremic syndrome relates toerythrocyte glycosphingolipid patterns., J Infect Dis, 168, 476.

EQUIVALENTS

[0220] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

What is claimed is:
 1. A hybrid molecule comprising a first domain and asecond domain covalently linked, wherein (a) said first domain comprisesa domain which is capable of specific binding to Gb₃; (b) said seconddomain comprising a moiety selected from the group consisting of drugmoiety, a nucleic acid, a probe, a polypeptide, and a hook, with theproviso that the second domain is not a verotoxin or a fragment thereof.2. The hybrid molecule of claim 1, wherein the first domain is averotoxin or a verotoxin subunit.
 3. The hybrid molecule of claim 1,wherein the first domain is VT-B.
 4. The hybrid molecule of claim 1,wherin the second domain is a polypeptide.
 5. The hybrid molecule ofclaim 4, wherein the polypeptide is a DNA binding element.
 6. The hybridmolecule of claim 1, wherein the second domain is a nucleic acid.
 7. Thehybrid molecule of claim 6, wherein the nucleic acid is an antisensenucleic acid.
 8. A pharmaceutical composition comprising the hybridmolecule of claim 1 and a pharmaceutically acceptable carrier.
 9. Amethod for modulating a cell-associated activity comprising contacting acell with the hybrid molecule of claim 1 such that a cell associatedactivity is altered relative to the cell-associated activity of the cellin the absence of the hybrid molecule.
 10. A method for directing thedelivery of the hybrid compound of claim 1 to a particular intracellularlocation in a cell, the method comprising contacting the cell with thehybrid compound, optionally in the presence of a compound which altersfatty acid composition of Gb₃, such that the hybrid compound isdelivered to a particular intracellular location in the cell.
 11. Use ofa hybrid compound of claim 1 for the manufacture of a medicament fortreatment, prophylaxis, or diagnosis.